The ability of nucleic acids to form sequence-specific hydrogen bonds or to hybridize with complementary strands of nucleic acids serves as the basis for techniques generally called hybridization assays.
In a hybridization assay, a nucleic acid having a selected sequence is used as a probe to search a sample for a target nucleic acid sequence which is complementary in sequence to the probe; binding of the two sequences is subsequently detected using a variety of known techniques. For example, labelling the hybridization complex formed by the probe and the target makes it possible to detect and, if desired, quantitate the target sequence in the sample.
In some hybridization assays, suitable pairs of a capture probe and a substance having an affinity for the capture probe are used to identify the hybridization complex (target nucleic acid sequence-probe complex) and/or remove it from the sample. For example, a poly- or oligonucleotide whose nucleic acid sequence is complementary to a region of the probe can be used. Suitable pairs of affinity component capture probe can be: poly(dG)-poly(dC) (polydioxriboguanylate-polydeoxyribocytidylate); poly(dA)-poly(dT) (polydeoxyriboadenylate-polydeoxyribothymidylate); and poly(dA)poly(U) (polydeoxyriboadenylate-polyuridylate). One member of this affinity pair can be affixed to a solid phase or solid support, such as a chromatography column, filter, plastic surface, glass etc.
Because all strains of a particular microorganism share a genetic component in the form of nucleic acids susceptible to diagnosis by means of a hybridization assay, such hybridization assays are valuable research medical tools. Detection of specific target nucleic acids make it possible to diagnose bacterial, fungal and vital disease states in humans, animals and plants. Additionally, the ability to probe for a specific nucleotide sequence is of potential use in the identification and diagnosis of human genetic disorders.
A particular disadvantage, however, of the use of nucleic acid hybridization schemes based on use of an affinity pair, such as dA-dT, is the possible interference with assay performance by naturally-occurring substances, such as molecules containing poly(rA) (polyriboadenylate) or poly(dA). Mammalian cells, which are found at various levels in virtually all clinical samples, especially blood, contain about 1 attomole (1.times.10.sup.-18 mole) of poly(rA) per cell (T. Maniatis et al., Molecular Cloning: A Laboratory Manual, p. 188 (1982)). They also contain about 100 ppm poly(dA) and poly(dT) by weight.
If present at high enough levels, endogenous poly(dA) and especially poly(rA), can compete with dA-containing probes for binding to supports having poly(dT). These compounds can also compete with any dA-tailed probes bound to the target molecule in solution. The result is a diminished assay signal and potentially higher backgrounds. The diminished signal results from reduced binding of probe: target complexes to the solid support. The higher backgrounds arise if the poly(rA)- and/or poly(dA)-containing sequences have a detectable amount of homology with the labeled probe. If the molecule to be detected is a poly(rA) mRNA or otherwise contains poly(rA), the specificity of the hybridization assay is reduced because binding of this molecule to the solid support occurs through the naturally-occurring poly(rA) and not through the highly specific poly(dA)-containing capture probe. Methods that prevent endogenous poly(rA) from binding to supports containing polydeoxyribothymidylic acid (poly(dT) are necessary to detect specific poly(rA)-containing mRNAs or other polyadenylated sequences.
There is presently no method available for effectively reducing,interference from endogenous poly(rA) and poly(dA) sequences in affinity pair hybridization assays. Methods employing finely divided particles can, in principle, block potential signal dimunition from poly(rA) and poly(dA) if enough solid phase is used that all of the target molecules and all of the endogenous poly(rA)- and poly(dA)-containing molecules bind to it. Collins, European Patent Application Number 0 265 244; Soderlund, UK Patent Application Number GB 2169403A; Stabinsky, U.S. Pat. No. 4,751,177. However, the use of such a quantity of solid phase particles can cause an unacceptable increase in the normal level of nonspecific background. Its use can also cause an increase in background if a small amount of labeled probe binds to non-target molecules containing poly(rA) or poly(dA).
These methods of reducing interference from endogenous polynucleotides are of limited utility when capture and detection are done on preferred supports for non-isotopic affinity-pair assays, such as poly(dT)-nitrocellulose and poly(dT)-polystyrene. These supports have only a limited binding capacity of about one microgram or less of polydeoxyriboadenylate(dA-12). M. Collins, European Patent Application No. 0 265 244. Furthermore, these supports can be saturated by poly(A)-containing mRNA present in about one million mammalian cells (based on the estimate of about 1 attomole of poly(rA)-200 per cell in Maniatis et al., supra). Although presently-available methods are useful for isotopic detection on magnetic beads, cellulose, and glass solid supports, nonisotopic affinity schemes on those solid supports have not shown sensitivity comparable to detection on poly(dT)-nitrocellulose and poly(dT)polystyrene. M. Collins, European Patent Application No. 0 265 244. In addition, there are no methods for the elimination of competition for binding to poly(dT) between endogenous poly(dA) and the dA-containing capture probes.
It would be useful to have a method whereby affinity-pair hybridization assays could be performed even in the presence in a sample of potentially interfering polymeric nucleotides and interference by the endogenous polynucleotides could be reduced.